Viral capsid proteins are important regulators of viral processes from translation, to RNA replication, to RNA packaging, and the modulation of cellular innate immune responses. In viruses with segmented genomes that are packaged into separate virions, the capsid protein must coordinate the activities for each of the RNAs. The overall goal of this project is to better understand the early steps in an RNA virus infection. That is, how does the capsid interact with the viral RNAs within the virion and facilitate entry into cells? Brome mosaic virus (BMV) will be the model virus for this study. BMV is a nonenveloped segmented positive-strand RNA virus that has served as a model system with notable contributions to the cis- and trans-acting factors, the formation of the replicase complex, and the mechanism of viral RNA-dependent RNA synthesis. Preliminary results showed that an arginine-rich motif of the BMV capsid contributes to the selective encapsidation of BMV RNAs and acts as a cell-penetration signal. The latter set of observations led to the hypothesis that plant viruses gain entry into cells through a protein transduction mechanism. This project contains two aims, the first will seek to understand the architecture of the virion through a combination of cryoelectron microscopy and single particle reconstruction, biochemical mapping of RNA conformation and RNA-capsid interaction, and a molecular genetic analysis of how the interactions will influence the infection process. The second aim seeks to elucidate the mechanism of cell entry of the cell-penetration peptides and the viral capsid using fluorescently-labeled peptides and viruses, confocal microscopy and electron microscopy. The work will lead to improved understanding of the viral infection process.